Xpressed in CRC and represented a potential effective predictor of poor prognosis in CRC patients, making it an attractive novel target for molecular imaging and therapy [30]. Several previous reports demonstrated that the VPAC1 receptor is highly expressed in CRC and plays a major role in the progression of CRC [11,14], making it one of the most promising novel candidate markers for early CRC detection. Therefore, the screening and identification of peptides that specifically bind to the VPAC1 receptor will aid the development of novel probes for CRC detection and 11967625 therapy. For this purpose, we utilized a phage display peptide library, and to the best of our knowledge, this is the first time that the VPAC1 receptor has been used as a target to screen a 12-mer phage display peptide library in an attempt to obtain peptides that specifically bind to the VPAC1 receptor. The most common screening strategy involves purifying a specific target, absorbing it to an affinity resin or ELISA plate, and screening for binding by adding a phage peptide library [31,32].Figure 4. Competitive inhibition of binding of the positive phage clone VP2 to CHO-K1/VPAC1 cells by the synthetic peptide VP2. The average inhibition rates at different concentrations of the VP2 peptide were shown. When the concentration of VP2 peptide was increased above 0.001 mg/ml, a significant inhibition occurred. An unrelated peptide displayed by the unrelated phage was used as a negative control. doi:10.1371/journal.pone.0054264.gScreening of a VPAC1-Binding JNJ-42756493 PeptideFigure 5. Binding specificity of the VP2 peptide to the VPAC1 receptor. (A) Competitive inhibition ELISA by VIP. The average inhibition rates at different concentrations of VIP were shown. When the concentration of VIP was increased above 0.001 mg/ml, a significant inhibition occurred. An unrelated peptide displayed by the unrelated phage was used as a negative control. (B) Flow cytometry analysis of the inhibition effect of VIP on binding of VP2 peptide to CHO-K1/VPAC1 cells. Here, a,d: blank control, b: VIP+FITC-VP2, e: Unrelated peptide+FITC-VP2, c,f: FITC-VP2. doi:10.1371/journal.pone.0054264.gThis method requires further confirmation of binding using the native form of the target in cells or tissues because peptides selected using purified recombinant protein might not be capable of accessing their targets in living cells. Our panning strategy employed intact and viable cells stably expressing VPAC1 receptors as target cells, which ensures a more specific target [33,34] and the isolation of a peptide that can specifically bind to cells expressing VPAC1 receptors. To decrease non-specific binding in each round, the original phage library was panned against absorber CHO-K1 cells before screening with CHO-K1/ VPAC1 cells. During the panning process, the temperature was set at 37uC when the phage library was incubated with the absorber CHO-K1 cells to ENMD-2076 chemical information ensure a minimal number of non-specific phages. When the phages were incubated with the target CHOK1/VPAC1 cells, the temperature was changed to 4uC to rigorously select internalized phages, which represents a deviation from many other panning procedures [24,26]. After four rounds of biopanning, the phage recovery rate gradually increased, and positive phage clones were 26001275 effectively enriched, while the phage input titer was maintained (Figure 2). Additionally, after acid elution in the fourth round, a specific elution with VIP wasperformed to recover as many specific posi.Xpressed in CRC and represented a potential effective predictor of poor prognosis in CRC patients, making it an attractive novel target for molecular imaging and therapy [30]. Several previous reports demonstrated that the VPAC1 receptor is highly expressed in CRC and plays a major role in the progression of CRC [11,14], making it one of the most promising novel candidate markers for early CRC detection. Therefore, the screening and identification of peptides that specifically bind to the VPAC1 receptor will aid the development of novel probes for CRC detection and 11967625 therapy. For this purpose, we utilized a phage display peptide library, and to the best of our knowledge, this is the first time that the VPAC1 receptor has been used as a target to screen a 12-mer phage display peptide library in an attempt to obtain peptides that specifically bind to the VPAC1 receptor. The most common screening strategy involves purifying a specific target, absorbing it to an affinity resin or ELISA plate, and screening for binding by adding a phage peptide library [31,32].Figure 4. Competitive inhibition of binding of the positive phage clone VP2 to CHO-K1/VPAC1 cells by the synthetic peptide VP2. The average inhibition rates at different concentrations of the VP2 peptide were shown. When the concentration of VP2 peptide was increased above 0.001 mg/ml, a significant inhibition occurred. An unrelated peptide displayed by the unrelated phage was used as a negative control. doi:10.1371/journal.pone.0054264.gScreening of a VPAC1-Binding PeptideFigure 5. Binding specificity of the VP2 peptide to the VPAC1 receptor. (A) Competitive inhibition ELISA by VIP. The average inhibition rates at different concentrations of VIP were shown. When the concentration of VIP was increased above 0.001 mg/ml, a significant inhibition occurred. An unrelated peptide displayed by the unrelated phage was used as a negative control. (B) Flow cytometry analysis of the inhibition effect of VIP on binding of VP2 peptide to CHO-K1/VPAC1 cells. Here, a,d: blank control, b: VIP+FITC-VP2, e: Unrelated peptide+FITC-VP2, c,f: FITC-VP2. doi:10.1371/journal.pone.0054264.gThis method requires further confirmation of binding using the native form of the target in cells or tissues because peptides selected using purified recombinant protein might not be capable of accessing their targets in living cells. Our panning strategy employed intact and viable cells stably expressing VPAC1 receptors as target cells, which ensures a more specific target [33,34] and the isolation of a peptide that can specifically bind to cells expressing VPAC1 receptors. To decrease non-specific binding in each round, the original phage library was panned against absorber CHO-K1 cells before screening with CHO-K1/ VPAC1 cells. During the panning process, the temperature was set at 37uC when the phage library was incubated with the absorber CHO-K1 cells to ensure a minimal number of non-specific phages. When the phages were incubated with the target CHOK1/VPAC1 cells, the temperature was changed to 4uC to rigorously select internalized phages, which represents a deviation from many other panning procedures [24,26]. After four rounds of biopanning, the phage recovery rate gradually increased, and positive phage clones were 26001275 effectively enriched, while the phage input titer was maintained (Figure 2). Additionally, after acid elution in the fourth round, a specific elution with VIP wasperformed to recover as many specific posi.